CN103087165A - Sodium hydrogen pump protein, and coding gene and applications thereof - Google Patents
Sodium hydrogen pump protein, and coding gene and applications thereof Download PDFInfo
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- CN103087165A CN103087165A CN2011103381872A CN201110338187A CN103087165A CN 103087165 A CN103087165 A CN 103087165A CN 2011103381872 A CN2011103381872 A CN 2011103381872A CN 201110338187 A CN201110338187 A CN 201110338187A CN 103087165 A CN103087165 A CN 103087165A
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- sodium hydrogen
- hydrogen pump
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- seq
- albumen
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Abstract
The invention relates to a sodium hydrogen pump protein with an amino acid sequence represented by SEQ ID No.2. Or, the sodium hydrogen pump protein has an amino acid sequence with sodium hydrogen pump protein activity, wherein the sequence is obtained by that the amino acid sequence represented SEQ ID No.2 is subjected to substitution, deletion, or addition of one or more amino acid residues. The invention also relates to a coding gene of the sodium hydrogen pump protein. The gene has a nucleotide sequence represented by SEQ ID No.1, or the gene has a nucleotide sequence coding the amino acid sequence represented by the SEQ ID No.2. The invention also relates to a recombinant vector and cells comprising the sodium hydrogen pump gene, and an application of the sodium hydrogen pump protein, the coding gene thereof, and the recombinant vector and the cell comprising the gene in culturing alkali-resistant genetically modified organisms.
Description
Technical field
The present invention relates to a kind of sodium hydrogen pump albumen, the encoding gene that also relates to sodium hydrogen pump albumen, and the recombinant vectors and the cell that contain sodium hydrogen pump gene, and sodium hydrogen pump albumen and encoding gene thereof contain the recombinant vectors of this gene and cell and have application in the alkali resistance genetically modified organism in cultivation.
Background technology
Sodium hydrogen pump (Na
+/ H
+Antiporter) be to be responsible for Na in cell
+/ H
+A kind of cross-film transport protein of exchange extensively is present in the film of the plasma membrane of bacterium, people and higher plant and many eukaryotic cells devices.Plasma membrane H
+-ATPase uses the energy of hydrolysising ATP H
+Pump cell from tenuigenin, produce the H across plasma membrane
+Gradient of electrochemical potential provides energy, drives the sodium hydrogen pump albumen on plasma membrane, makes H
+Enter cell along its electrochemical potential, simultaneously Na
+Contrary its electrochemical potential is discharged cell.Sodium hydrogen pump albumen passes through Na
+Efflux to keep intracellular low Na
+Stablizing of level and pH value is the saline and alkaline key factor of biomass cells tolerance, and it in the generation and ion balancing procedure of organoid, also carries out the function of volume and osmoregulation in addition, is the key factor that keeps the cell plasma equilibrium.
Summary of the invention
The present inventor is based on the molecular biology research to the sodium hydrogen pump, be surprised to find that from Alkaliphilic bacillus (Bacillus pseudofirmus OF4) and obtained a kind of sodium hydrogen pump albumen and encoding gene thereof, also provide on the other hand the recombinant vectors and the cell that contain sodium hydrogen pump gene, and sodium hydrogen pump albumen and encoding gene thereof contain the recombinant vectors of this gene and cell and have application in the alkali resistance genetically modified organism in cultivation.
The invention provides a kind of sodium hydrogen pump albumen, wherein, this sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2, and perhaps this sodium hydrogen pump albumen has and will still have the aminoacid sequence of sodium hydrogen pump protein-active after replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID No:2 through one or several amino-acid residue.
The present invention also provides a kind of sodium hydrogen pump gene, and wherein, this gene has the nucleotide sequence shown in SEQ ID No:1, and perhaps this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.
In addition, the present invention also provides a kind of recombinant vectors, and wherein, this recombinant vectors contains sodium hydrogen pump gene provided by the invention.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains sodium hydrogen pump gene provided by the invention.
The present invention also provides the sodium hydrogen pump albumen that obtains and encoding gene, the recombinant vectors that contains this gene and transgenic cell to have application in the alkali resistance genetically modified organism in cultivation.
Sodium hydrogen pump albumen has important application potential at aspects such as saline alkali tolerant plant cultivations.Obtain sodium hydrogen pump gene by the clone, and by genetically modified operation, microbe-derived sodium hydrogen pump gene is imported in vegetable cell, the transfer-gen plant that obtains the raising of salt tolerant alkalescence becomes possibility.
Description of drawings
Fig. 1 has shown the alkali-proof measurement result of the e. coli k12 (pUC18-Bpof4-NhaC) that recombinant vectors pUC18-Bpof4-NhaC imports, wherein, it is 8.0,8.5,9.0 and 9.5 (CAPS, the HEPES and the TRICINE that contain 50mM that the e. coli k12 (pUC18-Bpof4-NhaC) that recombinant vectors pUC18-Bpof4-NhaC is imported is inoculated into respectively pH, 5N NaOH adjusting) in LB liquid nutrient medium (100 μ g/ml penbritin), cultivate and measure OD after 12 hours
600, take the e. coli k12 that imports the pUC18 empty carrier as contrast (three every group parallel).
Fig. 2 has shown the intestinal bacteria KNabc of sodium hydrogen pump disappearance and the alkali-proof measurement result of KNabc (pUC18-Bpof4-NhaC) that imports recombinant vectors pUC18-Bpof4-NhaC, wherein, to be inoculated into respectively pH be 8.0,8.5,9.0 and 9.5 (CAPS, the HEPES and the TRICINE that contain 50mM with importing the KNabc (pUC18-Bpof4-NhaC) of recombinant vectors pUC18-Bpof4-NhaC and intestinal bacteria KNabc that the sodium hydrogen pump lacks, 5N NaOH adjusting) in LB liquid nutrient medium, cultivate and measure OD after 12 hours
600, take e. coli k12 as contrast (three every group parallel).
Embodiment
Following the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only is used for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of sodium hydrogen pump albumen, wherein, this sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2, and perhaps this sodium hydrogen pump albumen has and will still have the aminoacid sequence of sodium hydrogen pump protein-active after replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID No:2 through one or several amino-acid residue.Preferably, described sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2.
Correspondingly, the present invention also provides a kind of sodium hydrogen pump gene, and wherein, this gene has the nucleotide sequence shown in SEQ ID No:1, and perhaps this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.Preferably, described gene has the nucleotide sequence shown in SEQ ID No:1.
Sodium hydrogen pump gene provided by the invention is cloned from Alkaliphilic bacillus (Bacillus pseudofirmus OF4) and is obtained.
In addition, the present invention also provides a kind of recombinant vectors, and wherein, this recombinant vectors contains sodium hydrogen pump gene provided by the invention.
In the present invention, described " carrier " can select various carrier known in the art, as commercially available various plasmids, clay, phage and retrovirus etc.The preferred intestinal bacteria pUC18 of the present invention plasmid.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains sodium hydrogen pump gene provided by the invention.Described transgenic cell is prokaryotic cell prokaryocyte or eukaryotic cell, can be preferably intestinal bacteria, Bacillus subtilus or tobacco BY2 cell, most preferably intestinal bacteria.
In addition, the present invention also provides sodium hydrogen pump albumen provided by the invention and encoding gene thereof and has contained the recombinant vectors of sodium hydrogen pump gene and transgenic cell has application in the alkali resistance genetically modified organism in cultivation.Described biology is plant or microorganism.
Embodiment 1
The clone of the nucleotide sequence of coding sodium hydrogen pump
(1) extraction and the purifying of the total DNA of Alkaliphilic bacillus (Bacillus pseudofirmus OF4)
Get fresh wet thallus 20 grams of Alkaliphilic bacillus (Bacillus pseudofirmus OF4), be suspended from 10 milliliters of 50 mM/ls of Tris damping fluids (pH 8.0), add a small amount of N,O-Diacetylmuramidase and 8 milliliters 0.25 mM/l ethylenediamine tetraacetic acid (EDTA) (EDTA) (pH 8.0), placed 20 minutes in 37 ℃ after mixing, then add 2 milliliter of 10% sodium lauryl sulphate (SDS), placed 5 minutes for 55 ℃, use respectively equal-volume phenol, each extracting of chloroform once, get the supernatant solution of last extracting, add 2 times of volume ethanol, precipitation DNA.After the DNA that precipitates recovery is successively used 70 volume % ethanolic solns and absolute ethanol washing, gained DNA is dissolved in 0.5 milliliter of TE damping fluid, and (pH 8.0,10 mM/ls of Tris, 1 mM/l of EDTA), add 10 mg/ml RNA enzyme (RNase) 3 microlitres, 37 ℃ are incubated 1 hour, use respectively equal-volume phenol, each extracting of chloroform once, get supernatant liquor and add 2 times of volume ethanol, precipitation reclaims DNA, after successively using 70 volume % ethanolic solns and absolute ethanol washing, and vacuum-drying DNA precipitation, use deionized water dissolving, get total DNA solution.The ultraviolet spectrophotometer measurement result of DNA solution is A
260/ A
280=1.818, A
260/ A
230=2.052.
(2) sodium hydrogen pump gene cloning
After analyzing the genomic information of Alkaliphilic bacillus (Bacillus pseudofirmus OF4), the upstream and downstream primer of design sodium hydrogen pump gene, wherein upstream primer is 5 '-TATGACCATGATTACATGG AAAAAGAGAGAAAAC-3 ', and downstream primer is 5 '-CAGGTCGACTCTAGATCAA TCACACCAGAG-3 '.By the nucleic acid polymerase Pyrobest (Takara) of high-fidelity, go out the total length of sodium hydrogen pump gene as template amplification take Alkaliphilic bacillus (the Bacillus pseudofirmus OF4) genome of said extracted.By the size of 1% agarose gel electrophoresis testing goal gene, and the PCR product is delivered to the order-checking of Nuo Sai genome company, obtain at last the nucleotide sequence as shown in SED ID NO:1 of 663bp.
The gene function checking of embodiment 2 coding sodium hydrogen pumps
(1) structure of recombinant cloning vector pUC18-BpOF4-NhaC
Utilize Cycle-pure Kit purifying PCR product obtained above.Utilize Fast clone Kit that the PCR product of purifying is connected with pUC18, the recombinant vectors pUC18-Bpof4-NhaC that builds is imported in e. coli k12 BW25113 by chemical transformation.The screening of the LB flat board by containing 100 μ g/ml penbritins and PCR checking obtain containing the positive colony e. coli k12 (pUC18-Bpof4-NhaC) that recombinant vectors pUC18-Bpof4-NhaC inserts, and confirm that through order-checking the nucleotide sequence of extension increasing sequence that pUC18-Bpof4-NhaC inserts and sodium hydrogen pump is in full accord.
(2) structure of e. coli k12 BW25113 sodium hydrogen pump deletant
The primer amplification target practice gene that contains the DNA fragmentation of the upstream and downstream homology arm that remains to be knocked out gene by design utilizes intestinal bacteria to enter phage and has the λ Red recombination system different with the host goal gene is knocked out fast and accurately.By knocking out the sodium hydrogen pump gene of e. coli k12 BW2511, obtain the mutant E.coli KNabc that e. coli k12 BW25113 sodium hydrogen pump gene lacks fully, sodium hydrogen pump gene is replaced by kanamycin gene.
(3) the alkali-proof mensuration of the e. coli k12 (pUC18-Bpof4-NhaC) of recombinant vectors pUC18-Bpof4-NhaC importing
It is 8.0,8.5,9.0 and 9.5 (CAPS, the HEPES and the TRICINE that contain 50mM that the e. coli k12 (pUC18-Bpof4-NhaC) that recombinant vectors pUC18-Bpof4-NhaC is imported is inoculated into respectively pH, 5N NaOH adjusting) in LB liquid nutrient medium (100g/ml penbritin), cultivate and measure OD after 12 hours
600, take the e. coli k12 that imports the pUC18 empty carrier as contrast (three every group parallel), result as shown in Figure 1.
(4) the alkali-proof mensuration of KNabc (pUC18-BpOF4-NhaC) of the intestinal bacteria KNabc of sodium hydrogen pump disappearance and importing recombinant vectors pUC18-BpOF4-NhaC
To be inoculated into respectively pH be 8.0,8.5,9.0 and 9.5 (CAPS, the HEPES and the TRICINE that contain 50mM with importing the KNabc (pUC18-Bpof4-NhaC) of recombinant vectors pUC18-Bpof4-NhaC and intestinal bacteria KNabc that the sodium hydrogen pump lacks, 5N NaOH adjusting) in LB liquid nutrient medium, cultivate and measure OD after 12 hours
600, take e. coli k12 as contrast (three every group parallel), result as shown in Figure 2.
As can be seen from Figure 1, the e. coli k12 that imports recombinant vectors pUC18-Bpof4-NhaC is in 8.0,8.5,9.0 and 9.5 LB liquid nutrient medium (100 μ g/ml penbritin) at pH, measures OD after 12 hours
600Apparently higher than the e. coli k12 that imports the pUC18 empty carrier, this has proved that also the importing of sodium hydrogen pump gene (Bpof4-NhaC) can improve the alkali resistance of e. coli k12.
As can be seen from Figure 2, import the intestinal bacteria KNabc of recombinant vectors pUC18-Bpof4-NhaC at pH 8.0,8.5, in the LB liquid nutrient medium of 9.0 and 9.5 (containing 50mM CAPS, HEPES and TRICINE, 5N NaOH adjusting), measure OD after 12 hours
600Apparently higher than the deletant KNabc that imports intestinal bacteria sodium hydrogen pump, this has proved that also the importing of sodium hydrogen pump gene (Bpof4-NhaC) can improve the alkali resistance of intestinal bacteria KNabc.
In sum, the sodium hydrogen pump gene that provides of the application has important application potential at the aspects such as cultivation of Salt And Alkali Tolerance biology.Obtain sodium hydrogen pump gene by the clone, and by genetically modified operation, microbe-derived sodium hydrogen pump is imported in vegetable cell, the transfer-gen plant that obtains the raising of salt tolerant alkalescence becomes possibility.
Claims (9)
1. sodium hydrogen pump albumen, it is characterized in that, this sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2, and perhaps this sodium hydrogen pump albumen has and will still have the aminoacid sequence of sodium hydrogen pump protein-active after replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID No:2 through one or several amino-acid residue.
2. sodium hydrogen pump albumen according to claim 1, wherein, this albumen has the aminoacid sequence shown in SEQ ID No:2.
3. a sodium hydrogen pump gene, is characterized in that, this gene has the nucleotide sequence shown in SEQ ID No:1, and perhaps this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.
4. gene according to claim 3, wherein, this gene has the nucleotide sequence shown in SEQ ID No:1.
5. a recombinant vectors, is characterized in that, this recombinant vectors contains gene claimed in claim 3.
6. a transgenic cell, is characterized in that, this transgenic cell contains gene claimed in claim 3.
7. transgenic cell according to claim 6, wherein, described transgenic cell is prokaryotic cell prokaryocyte or eukaryotic cell.
8. sodium hydrogen pump albumen claimed in claim 1, gene claimed in claim 3, recombinant vectors claimed in claim 5, transgenic cell claimed in claim 6 have application in the alkali resistance genetically modified organism in cultivation.
9. application according to claim 8, wherein, described biology is plant or microorganism.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1454997A (en) * | 2003-01-09 | 2003-11-12 | 复旦大学 | Rape sodium-hydrogen pump transport protein coding sequence and application thereof |
| CN1958796A (en) * | 2005-10-31 | 2007-05-09 | 中国科学院微生物研究所 | Nucleotide sequence protein gene of coded natrium hydrogen pump, and application |
| US20090258401A1 (en) * | 2005-12-16 | 2009-10-15 | Mayu Iyo | L-amino acid producing bacterium and method of producing l-amino acid |
-
2011
- 2011-10-31 CN CN2011103381872A patent/CN103087165A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1454997A (en) * | 2003-01-09 | 2003-11-12 | 复旦大学 | Rape sodium-hydrogen pump transport protein coding sequence and application thereof |
| CN1958796A (en) * | 2005-10-31 | 2007-05-09 | 中国科学院微生物研究所 | Nucleotide sequence protein gene of coded natrium hydrogen pump, and application |
| US20090258401A1 (en) * | 2005-12-16 | 2009-10-15 | Mayu Iyo | L-amino acid producing bacterium and method of producing l-amino acid |
Non-Patent Citations (2)
| Title |
|---|
| JANTO B ET AL: "EBI accession no.UNIPROT：D3FPS6", 《UNIPROT》 * |
| TAKAMI H ET AL: "EBI accession no.UNIPROT：Q9K5Y9", 《UNIPROT》 * |
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Application publication date: 20130508 |